Functional and Mechanistic Studies of Two Anti-coccidial Herbs, Bidens pilosa and Artemisia indica.

Currently, antibiotics are commonly used to treat coccidiosis, a severe protozoal disease in chickens. However, due to growing concerns about the antibiotic residue in meat and eggs, phytogenic formulations are becoming an attractive approach to manage this disease. In this study, we investigated the anti-coccidial function and mechanism of phytogenic formulations composed of Bidens pilosa, Artemisia indica, and both used in combination. We found that these formulations increased the survival rate and reduced body weight loss, the feed conversion ratio, oocyst excretion, bloody stools, and gut lesions of chickens. Mechanistic studies showed that A. indica, but not B. pilosa, reduced the survival of Eimeria oocysts. Accordingly, they both inhibited oocyst sporulation and sporozoite invasion into Madin-Darby bovine kidney (MDBK) cells. Overall, we demonstrate that these formulations protect chickens against coccidiosis. Moreover, a combination of B. pilosa and A. indica has an additive effect on coccidiosis control and growth performance in chickens compared to either one used alone.

First report of youcai mosaic virus in firecracker flower (Crossandra infundibuliformis) in Taiwan.

Firecracker flower or crossandra (Crossandra infundibuliformis), an ornamental native to southern Asia, is commonly grown as bedding plants in the garden. In January 2021, crossandra plants showing mosaic, chlorotic ringspot, and leaf deformation (Suppl. Fig. 1) were observed at a recreational farm in Zhuolan Township (Miauli County, Taiwan) (E120°82'62'', N24°33'30'') . Transmission electron microscope (JEM-1400, JOEL, Japan) examination by negative staining of 10 affected plants indicated the presence of particles resembling a tobamovirus in all examined affected samples. However, no tobamovirus-like particles were observed in the crude sap prepared from healthy crossandra leaf tissues. Total RNA was extracted from affected leaves and used for RT-PCR amplification using the tobamovirus group-specific primer pair Tob Uni1 (5'-ATTTAAGTGGAGGGAAAACCACT-3') and Tob Uni2 (5'-GTYGTTGATGAGTTCGTGGA-3') (Letschert et al., 2002). A cDNA fragment of about 650-bp was amplified and Sanger sequenced (ABI PRISM 3730 DNA Sequencer, Biotechnology Center at National Chung Hsing University, Taichung, Taiwan ), revealing 98% sequence identity to that of a Brassica isolate of youcai mosaic virus (YoMV, AY318866). The virus was isolated through mechanical inoculation onto Chenopodium quinoa to yield two pure isolates, designated FC-1 and FC-2. Mechanical inoculation of FC-1 and FC-2 back to virus-free Crossandra infundibuliformis plants (5 for each isolate) resulted in systemic mosaic, chlorotic ringspot, and leaf deformation. All mock and healthy controls were symptomless and failed to obtain any RT-PCR products with YoMV-specific primers CPF1 (5'- ATGGTTTACAACATCACGAG-3') and CPR1 (5'-CTATGTAGCTGGCGCAGTAG-3'). Systemic symptoms of mild mosaic, ringspot, leafroll, and necrosis appeared on some of the tobaccos (Nicotiana benthamiana, N. tabacum, N. rustica), and cruciferous vegetables (Brassica rapa subsp. chinensis cv. Known-You No.2), B. rapa subsp. pekinensis cv. Autumn Sun), B. oleracea var. italica cv. Ching Hua), B. oleracea var. capitata cv. Green Peak), and Raphanus sativus cv. Snow Lady). However, inoculation of FC-1 and FC-2 on pepper (Capsicum annuum cv. Blue Star) resulted in severe necrosis on leaves and necrotic sunken spots on petioles and stems causing acute wilting and quick death. In stark contrast, FC-1 and FC-2 only induced local lesions on inoculated leaves of Chenopodium quinoa, Gomphrena globose, and Carica papaya. The infectivity of FC isolates to all plants used in host range tests were further confirmed by RT-PCR as mentioned. Oligonucleotide primers (Suppl. Table 1) specifically complementary to YoMV sequence were designed and used to amplify full-length genomic sequences of FC-1 and FC-2 isolates by RT-PCR and Sanger sequencing. Sequence analysis revealed that the genome of both FC-1 and FC-2 isolates consists of 6302 nucleotides. The viral genome has four open reading frames encoding a small replicase subunit, a RNA-dependent RNA polymerase , a movement protein, and a coat protein (CP), respectively. Both sequences, which shared 99.6% identity with each other, have been deposited in the NCBI database (Genbank Accession Numbers LC701592 and LC701593). FC-1 and FC-2 and the deduced amino acid sequences of CP shared 91.2% - 98.9% and 93.4 - 99.4% similarities, respectively, to those of published YoMV strains, confirming the identity of FC-1 and FC-2. RT-PCR analyses detected YoMV in all (100%) crossandra samples collected from the field (Suppl. Fig. 2). YoMV, formerly named Chinese rape mosaic virus (CRMV) or oilseed rape mosaic virus (ORMV) (Zhu et al., 2001), has been reported to infect cruciferous, solanaceous, and ornamental crops in Asia and Europe (Ju et al., 2019). The firecracker flower is a common and popular ornamental in Taiwan, even though its economic values are not so important. However, finding YoMV in firecracker flower may have epidemiological impacts as YoMV can infect economically important cruciferous and solanaceous crops. To our knowledge, this is the first report of YoMV infecting firecracker flowers in Taiwan.

Rose Virome Analysis and Identification of a Novel Ilarvirus in Taiwan.

Rose (Rosa spp.), especially R. hybrida, is one of the most popular ornamental plants in the world and the third largest cut flower crop in Taiwan. Rose mosaic disease (RMD), showing mosaic, line patterns and ringspots on leaves, is a common rose disease caused by the complex infection of various viruses. Due to pests and diseases, the rose planting area in Taiwan has been decreasing since 2008; however, no rose virus disease has been reported in the past five decades. In the spring of 2020, rose samples showing RMD-like symptoms were observed at an organic farm in Chiayi, central Taiwan. The virome in the farm was analyzed by RNA-seq. Rose genomic sequences were filtered from the obtained reads. The remaining reads were de novo assembled to generate 294 contigs, 50 of which were annotated as viral sequences corresponding to 10 viruses. Through reverse transcription-polymerase chain reaction validation, a total of seven viruses were detected, including six known rose viruses, namely apple mosaic virus, prunus necrotic ringspot virus, rose partitivirus, apple stem grooving virus, rose spring dwarf-associated virus and rose cryptic virus 1, and a novel ilarvirus. After completing the whole genome sequencing and sequence analysis, the unknown ilarvirus was demonstrated as a putative new species, tentatively named rose ilarvirus 2. This is the first report of the rose virus disease in Taiwan.

Adverse pulmonary impacts of environmental concentrations of oil mist particulate matter in normal human bronchial epithelial cell.

Airborne oil mist particulate matter (OMPM) is generated during industrial processes such as metalworking and may be associated with pulmonary dysfunction. In this study, we employed the normal human bronchial epithelial BEAS-2B cell line to elucidate the association between pulmonary toxicity and OMPM of 2.5-10 µm, 1.0-2.5 µm and <1.0 µm particle sizes (OMPM10-2.5, OMPM2.5-1.0 and OMPM1.0). We measured OMPM concentrations at a precision machinery factory to estimate lung deposition rates and select realistic environmental concentrations for testing. All OMPMs (1-50 µg/cm2) significantly decreased BEAS-2B cell viability (>38% of control), except for low-dose OMPM1.0 (1 µg/cm2). OMPM10-2.5 and OMPM2.5-1.0, but not OMPM1.0, induced oxidative stress (1.5-4-fold increase compared with the control) and increased the production of proinflammatory cytokines (1.5-3-fold). However, only OMPM1.0 induced pulmonary epithelial barrier dysfunction via depletion of zonula occludens (0.65-0.8-fold) and α1-antitrypsin proteins (0.65-0.8-fold). In conclusion, a higher risk of lung disease was associated with smaller particle size OMPM. Exposure to OMPM1.0 may be a potential risk factor for chronic obstructive pulmonary disease. The evidence also demonstrates that occupational exposure to OMPM may cause pulmonary disease at very low concentrations.

Helicobacter pylori infection generated gastric cancer through p53-Rb tumor-suppressor system mutation and telomerase reactivation.

AIM: To investigate the relationship between Helicobacter pylori (H.pylori) infection and the expressions of the p53, Rb, c-myc, bcl-2 and hTERT mRNA in a series of diseases from chronic gastritis (CG), intestinal metaplasia type I or II(IMI-II), intestinal metaplasia type III (IMIII), mild or modest dysplasia (DysI-II), severe dysplasia (DysIII) to gastric cancer(GC) and to elucidate the mechanism of gastric carcinogenesis relating to H.pylori infection. METHODS: 272 cases between 1998 and 2001 were available for the study including 42 cases of CG, 46 cases of IMI-II, 25 cases of IMIII, 48 cases of DysI-II, 27 cases of DysIII, 84 cases of GC. H.pylori infection and the expressions of p53, Rb, c-myc, bcl-2 were detected by means of streptavidin-peroxidase (SP) immunohistochemical method. HTERT mRNA was detected by in situ hybridization (ISH). RESULTS: The expressions of p53, Rb, c-myc, hTERT mRNA and bcl-2 were higher in the GC than in CG, IM, Dys. The expression of c-myc was higher in IMIII with H.pylori infection (10/16) than that without infection (1/9) and the positive rate in DysI-II and DysIII with H.pylori infection was 18/30 and 13/17, respectively, higher than that without infection (4/18 and 3/10, respectively). In our experiment mutated p53 had no association with H.pylori infection, the expression of Rb was associated with H.pylori infection in GC, but the p53-Rb tumor-suppressor system abnormal in DysI-II cases, DysIII and GC cases with H.pylori infection was 21/30, 15/17 and 48/48 respectively, higher than non-infection groups (4/18, 3/10, 28/36). Furthermore the level of hTERT mRNA in GC with H.pylori infection (47/48) was higher than that without infection (30/36), however the relationship between bcl-2 and H.pylori was only in IMIII. C-myc had a close association with hTERT mRNA in DysIII and GC (P=0.0 253,0.0 305 respectively). CONCLUSION: In the gastric carcinogenesis, H.pylori might cause the severe imbalance of proliferation and apoptosis in the precancerous lesions (IMIII and GysIII) first, leading to p53-Rb tumor-suppressor system mutation and telomerase reactivation, and finally causes gastric cancer.